The following is a brief description of the delivery of biopolymers. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Trafficking of large, charged molecules into living cells is highly restricted by the complex membrane systems of the cell. Specific tranporters allow the selective entry of nutrients or regulatory molecules, while excluding most exogenous molecules such as nucleic acids and proteins. The two major strategies for improving the transport of foreign nucleic acids into cells are the use of viral vectors or cationic lipids and related cytofectins. Viral vectors can be used to transfer genes efficiently into some cell types, but they cannot be used to introduce chemically synthesized molecules into cells. An alternative approach is to use delivery formulations incorporating cationic lipids, which interact with nucleic acids through one end and lipids or membrane systems through another (for a review see Felgner, 1990, Advanced Drug Delivery Reviews, 5,162-187; Felgner 1993, J. Liposome Res., 3,3-16). Synthetic nucleic acids as well as plasmids may be delivered using the cytofectins, although their utility is often limited by cell-type specificity, requirement for low serum during transfection, and toxicity.
Since the first description of liposomes in 1965, by Bangham (J. Mol. Biol. 13, 238-252), there has been a sustained interest and effort in the area of developing lipid-based carrier systems for the delivery of pharmaceutically active compounds. Liposomes are attractive drug carriers since they protect biological molecules from degradation while improving their cellular uptake.
One of the most commonly used classes of liposomes formulation for delivering polyanions (e.g., DNA) are those that contain cationic lipids. Lipid aggregates can be formed with macromolecules using cationic lipids alone or including other lipids and amphiphiles such as phosphatidylethanolamine. It is well known in the art that both the composition of the lipid formulation as well as its method of preparation have effect on the structure and size of the resultant anionic macromolecule-cationic lipid aggregate. These factors can be modulated to optimize delivery of polyanions to specific cell types in vitro and in vivo. The use of cationic lipids for cellular delivery of biopolymers have several advantages. The encapsulation of anionic compounds using cationic lipids is essentially quantitative due to electrostatic interaction. In addition, it is believed that the cationic lipids interact with the negatively charged cell membranes initiating cellular membrane transport (Akhtar et al., 1992, Trends Cell Bio., 2, 139; Xu et al., 1996, Biochemistry 35, 5616).
The transmembrane movement of negatively charged molecules such as nucleic acids may therefore be markedly improved by coadministration with cationic lipids or other permeability enhancers (Bennett et al., 1992 Mol. Pharmacol., 41, 1023-33; Capaccioli et al., 1993, BBRC, 197,818-25; Ramila et al., 1993 J. Biol. Chem., 268,16087-16090; Stewart et al., 1992, Human Gene Therapy, 3, 267-275). Since the introduction of the cationic lipid DOTMA and its liposomal formulation Lipofectin(trademark) (Felgner et al., 1987, PNAS 84, 7413-7417; Eppstein et al., U.S. Pat. No. 4,897,355), a number of other lipid-based delivery agents have been described primarily for transfecting mammalian cells with plasmids or antisense molecules (Rose, U.S. Pat. No. 5,279,833; Eppand et al. U.S. Pat. No. 5,283,185; Gebeyehu et al., U.S. Pat. No. 5,334,761; Nantz et al., U.S. Pat. No. 5,527,928; Bailey et al., U.S. Pat. No. 5,552,155; Jesse, U.S. Pat. No. 5,578,475). However, each formulation is of limited utility because it can deliver plasmids into some but not all cell types, usually in the absence of serum (Bailey et al., 1997, Biochemistry, 36, 1628). Concentrations (charge and/or mass ratios) that are suitable for plasmid delivery (xcx9c5,000 to 10,000 bases in size) are generally not effective for oligonucleotides such as synthetic ribozymes or antisense molecules (xcx9c10 to 50 bases). Also, recent studies indicate that optimal delivery conditions for antisense oligonucleotides and ribozymes are different, even in the same cell type. However, the number of available delivery vehicles that may be utilized in the screening procedure is highly limited, and there continues to be a need to develop transporters that can enhance nucleic acid entry into many types of cells.
Epstein et al., U.S. Pat. No. 5,208,036, disclose a liposome, LIPOFECTIN(trademark), that contains an amphipathic molecule having a positively charged choline head group (water soluble) attached to a diacyl glycerol group (water insoluble). Promega (Wisconsin) markets another cationic lipid, TRANSFECTAM(trademark), which can help introduce nucleic acid into a cell.
Wagner et al., 1991, Proc. Nat. Acad. Sci. USA 88, 4255; Cotten et al., 1990, Proc. Nat. Acad. Sci. USA 87, 4033; Zenke et al., 1990, Proc. Nat. Acad. Sci. USA 87, 3655; and Wagner et al., Proc. Nat. Acad. Sci. USA 87, 3410, describe transferrin-polycation conjugates which may enhance uptake of DNA into cells. They also describe the feature of a receptor-mediated endocytosis of transferrin-polycation conjugates to introduce DNA into hematopoietic cells.
Wu et al., J. Biol. Chem. 266, 14338, describe in vivo receptor-mediated gene delivery in which an asialoglycoprotein-polycation conjugate consisting of asialoorosomucoid is coupled to poly-L-lysine. A soluble DNA complex was formed capable of specifically targeting hepatocytes via asialoglycoprotein receptors present on the cells.
Biospan Corporation International PCT Publication No. WO 91/18012, describe cell internalizable covalently bonded conjugates having an xe2x80x9cintracellularly cleavable linkagexe2x80x9d such as a xe2x80x9cdisulfide cleavable linkagexe2x80x9d or an enzyme labile ester linkage.
Brigham, U.S. Pat. No. 5,676,954 describes a method for the expression of nucleic acid following transfection into a target organ consisting of mammalian cells.
The references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the delivery vehicles of the instant invention.
This invention features cationic lipid-based compositions to facilitate delivery of negatively charged molecules into a biological system such as animal cells. The present invention discloses the design, synthesis, and cellular testing of novel agents for the delivery of negatively charged molecules in vitro and in vivo. Also disclosed are screening procedures for identifying the optimal delivery vehicles for any given nucleic acid and cell type. In general, the transporters described here were designed to be used either individually or as part of a multicomponent system. The compounds of the invention generally shown in FIG. 1, are expected to improve delivery of negatively charged molecules into a number of cell types originating from different tissues, in the presence or absence of serum.
The xe2x80x9cnegatively charged moleculesxe2x80x9d are meant to include molecules such as nucleic acid molecules (e.g., RNA, DNA, oligonucleotides, mixed polymers, peptide nucleic acid, and the like), peptides (e.g., polyaminoacids, polypeptides, proteins and the like), nucleotides, pharmaceutical and biological compositions, that have negatively charged groups that can ion-pair with the positively charged head group of the cationic lipids of the invention
In a first aspect the invention features a cationic lipid having the formula I: 
wherein, n is 1, 2 or 3 carbon atoms; n, is 2, 3, 4 or 5 carbon atoms; R and R1 independently represent C12-C22 alkyl chain which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and R2 and R3 are independently H, acyl, alkyl, carboxamidine, aryl, acyl, substituted carboxamidine, polyethylene glycol (PEG) or a combination thereof.
In a second aspect the invention features a cationic lipid having the formula II: 
wherein, n is 1, 2 or 3 carbon atoms; n, is 2, 3, 4 or 5 carbon atoms; R and R independently represent C12-C22 alkyl chain which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and Alk represents methyl, hydroxyalkyl (e.g., hydroxymethyl and hydroxyalkyl) or a combination thereof.
In a third aspect the invention features a cationic lipid having the formula III: 
wherein, R and R1 independently represent C12-C22 alkyl chain which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and R2 is H, PEG, acyl or alkyl.
In a fourth aspect the invention features a cationic lipid having the formula IV: 
wherein, n is 1-6 carbon atoms; R and R1 independently represent C12-C22 alkyl chain which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and R2 is H, carboxamidine, alkyl, acyl, aryl, PEG, substituted carboxamidine 
where R3 is H, or PO3 H2 and R4 is OH, NH2 or xe2x95x90O.
In a fifth aspect the invention features a cationic lipid having the formula V: 
wherein, n is 1-6 carbon atoms; X and X1 independently represent C12-C22 alkyl chain which are saturated or unsaturated wherein the unsaturation is represented by 1-4 double bonds, B is a nucleic acid base or H; and R5 is H, PEG, or carboxamidine.
In a sixth aspect the invention features a cationic lipid having the formula VI: 
wherein, n is 1, 2 or 3 carbon atoms; R and R1 independently represent C12-C22 alkyl chain which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds; and R2 and is independently H, polyethylene glycol (PEG) or 
For the above-referenced formulae, N, O, and H are Nitrogen, Oxygen and Hydrogen, according to the abbreviations well-known in the art.
In a seventh aspect the invention features a cationic lipid having the formula VII:
R6xe2x80x94L1-Cholesterol
wherein, R6 is selected from the group consisting of arginyl methyl ester, arginyl amide, homoarginyl methyl ester, homoarginyl amide, ornithine methyl ester, ornithine amide, lysyl methyl ester, lysyl amide, triethylenetetramine (TREN), N,Nxe2x80x2-di-carboxamidine TREN, N-benzyl histidyl methyl ester, pyridoxyl and aminopropylimidazole. L1 is a linker represented by R7PO2, wherein R7 is H, CH3, or CH2CH3. Examples of this group of compounds are: PH55933, PH55938, PH55939, PH55941, PH55942, PH55943 and PH55945.
In an eighth aspect the invention features a cationic lipid having the formula VIII:
R8xe2x80x94L2-Cholesterol
wherein, R8 is selected from the group consisting of arginyl, N-Boc arginyl, homoarginyl, N-Boc homoarginyl ornithine, N-Boc ornithine, N-benzyl histidyl, lysyl, N-Boc lysyl, N-methyl arginyl, N-methyl guanidine, guanidine and pyridoxyl. Is is a linker represented by NH, glycine, N-butyldiamine or guanidine. Examples of this compound is Boc arginine cholesterol amide (DS46596), N-guanyl-cholesterylamide (DS57511).
In a ninth aspect the invention features a cationic lipid having the formula IX: 
Wherein R is independently a C12-C22 alkyl chain which are saturated or unsaturated, wherein the unsaturation is represented by 1-4 double bonds and R1 is represented by TREN, N,Nxe2x80x2-di-carboxamidine TREN, lysyl, arginyl, ornithyl, homoarginyl, histidyl, aminopropylimidazole, spermine carboxylic acid.
By xe2x80x9cHead Groupxe2x80x9d is meant an amino-containing moiety that is positively charged and is capable of forming ion pairs with negatively charged regions of biopolymers such as nucleic acid molecules.
By xe2x80x9clipophilic groupxe2x80x9d is meant a hydrophobic lipid-containing group that facilitates transmembrane transport of the cationic lipid.
By xe2x80x9clinkerxe2x80x9d is meant a 1-6 atom carbon chain that links the head group with the lipophylic group.
By xe2x80x9cion pairxe2x80x9d is meant non-covalent interaction between oppositely charged groups.
Specifically, an xe2x80x9calkylxe2x80x9d group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxy, cyano, alkoxy, NO2 or N(CH3)2, amino, or SH.
By xe2x80x9calkoxyxe2x80x9d is meant an OR group, wherein R is an alkyl.
An xe2x80x9carylxe2x80x9d group refers to an aromatic group which has at least one ring having a conjugated xcfx80 electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) on aryl groups are halogen, trihalomethyl, hydroxyl, SH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.
The term xe2x80x9calkenylxe2x80x9d group refers to unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, NO2, halogen, N(CH3)2, amino, or SH.
The term xe2x80x9calkynylxe2x80x9d refers to an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, xe2x95x90O, xe2x95x90S, NO2 or N(CH3)2, amino or SH.
An xe2x80x9calkylarylxe2x80x9d group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above).
xe2x80x9cCarbocyclic arylxe2x80x9d groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted.
xe2x80x9cHeterocyclic arylxe2x80x9d groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
By xe2x80x9cacylxe2x80x9d is meant xe2x80x94C(O)R groups, wherein R is an alkyl or aryl.
In a preferred embodiment, the invention features process for the synthesis of the compounds of formula I-IX.
In another preferred embodiment a series of multidomain cellular transport vehicles (MCTV) including one or more lipids of formula I-IX that enhance the cellular uptake and transmembrane permeability of negatively charged molecules in a variety of cell types are described. The lipids of the invention are used either alone or in combination with other compounds with a neutral or a negative charge including but not limited to neutral lipid and/or targeting components, to improve the effectiveness of the lipid formulation in delivering and targeting the negatively charged polymers to cells. Another object is to describe the utility of these delivery vehicles for increasing the transport of other impermeable and/or lipophilic compounds into cells.
By xe2x80x9ccompounds with neutral chargexe2x80x9d is meant compositions which are neutral or uncharged at neutral or physiological pH. Examples of such compounds are cholesterol (i.e., a steroidal alcohol, as defined in Lehninger, Biochemistry, 1982 ed., Worth Pub., p. 315) and other steroids, cholesteryl hemisuccinate (CHEMS), dioleoyl phosphatidyl choline, distearoylphosphotidyl choline (DSPC), fatty acids such as oleic acid, phosphatidic acid and its derivatives, phosphatidyl serine, polyethylene glycol-conjugated phosphatidylamine, phosphatidylcholine, phosphatidylethanolamine and related variants, prenylated compounds including farnesol, polyprenols, tocopherol, and their modified forms, diacylsuccinyl glycerols, fusogenic or pore forming peptides, dioleoylphosphotidylethanolamine (DOPE), ceramide and the like.
Targeting components include ligands for cell surface receptors including, peptides and proteins, glycolipids, lipids, carbohydrates, and their synthetic variants.
In yet another preferred embodiment, the cationic lipid molecules of the invention are provided as a lipid aggregate, such as a liposome, and co-encapsulated with the negatively charged polymer to be delivered. Liposomes, which may be unilamellar or multilamellar, can introduce encapsulated material into a cell by different mechanisms. See, Ostro, Scientific American 102, January 1987. For example, the liposome can directly introduce its encapsulated material into the cell cytoplasm by fusing with the cell membrane. Alternatively, the liposome can be compartmentalized into an acidic vacuole (i.e., an endosome) having a pH below 7.0. This low pH allows ion-pairing of the encapsulated enhancers and the negatively charged polymer, which facilitates diffusion of the enhancer:polymer complex out of the liposome, the acidic vacuole, and into the cellular cytoplasm.
By xe2x80x9clipid aggregatexe2x80x9d is meant a lipid-containing composition (i.e., a composition comprising a lipid according to the invention) wherein the lipid is in the form of a liposome, micelle (non-lamellar phase) or other aggregates with one or more lipids.
In yet another preferred embodiment the invention features a lipid aggregate formulation including phosphatidylcholine (of varying chain length; e.g., egg yolk phosphatidylcholine), cholesterol, a cationic lipid, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polythyleneglycol-2000 (DSPE-PEG2000). The cationic lipid component of this lipid aggregate can be any cationic lipid known in the art such as dioleoyl 1,2,-diacyl-3-trimethylammonium-propane (DOTAP). In a preferred embodiment this cationic lipid aggregate comprises a cationic lipid described in any of the Formula I-IX.
In yet another preferred embodiment, polyethylene glycol (PEG) is covalently attached to the cationic lipids of the present invention. The attached PEG can be any molecular weight but is preferably between 2000-5000 daltons.
The molecules and methods of the present invention are particularly advantageous for introducing nucleic acid molecules into a cell. For example, the invention can be used for ribozyme delivery where its target site of action exists intracellularly.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.